Articulo

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2018 Jun; 162(2):99-106.
Dietary sucrose regulates the expression of the Cd36 gene in hepatic tissue
of rats with obesity and Non Alcoholic Fatty Liver Disease (NAFLD)
Rodolfo Quintana Castroa,b, Ida Soto Rodrigueza, Rosa A. Deschamps Lagoa, Peter Grube Pagolac, Jorge Rodriguez Antolind,
Adriana Peres Quintala, Jaime Rivera Riveraa, Alfonso Alexander Aguileraa,b
Aim. To evaluate the mRNA expression levels of Cd36 in adipose and hepatic tissues, in rats with NAFLD after the
consumption of sucrose for 10 and 20 weeks.
Methods. Twenty Wistar rats, all nearly 21 days old were divided into two experimental groups (NAFLD-10 and
­NAFLD-20), that received a standard diet (2014 Teklad Global) plus 30% sucrose in their drinking water for 10 and 20
weeks and the control groups (C Groups). Variables such as body weight, food intakes, and serum parameters were
measured. Adipose and hepatic tissues were extirpated; some tissue was preserved in formalin and some at -70 °C until
analysis. Histological analysis was carried out, and the Cd36 mRNA expression levels were determined.
Results. The rats in the NAFLD-10 and NAFLD-20 groups showed a significative increase in abdominal fat, triglycerides,
free fatty acids, insulin, AST, ALT, uric acid and HOMA index; as well as changes in the cellular dynamics in adipose
tissue, (adipocytes hypertrophic: >1500 µm2) with respect to the control groups (P<0.05). The histological analysis
showed development of mild portal hepatitis in rats of the NAFLD-10 group and grade 1 hepatic steatosis with mild
portal inflammation in rats of the NAFLD-20 group. Finally, Cd36 mRNA expression levels were significantly increased
in hepatic tissue after 10 (1.5-fold) and 20 (3.5-fold) weeks of sucrose ingestion (P<0.05).
Conclusion. mRNA expression is a molecular mechanism involved in the development of NAFLD associated with
obesity in rats consuming sucrose. However, there was increased Cd36 mRNA expression only in hepatic tissue while
in hypertrophic adipose tissue mRNA levels remained unchanged.
Key words: fatty liver, metabolic syndrome, sucrose, CD36, insulin resistance, obesity
Received: September 11, 2017; Accepted: April 4, 2018; Available online: May 15, 2018
https://doi.org/10.5507/bp.2018.016
Facultad de Bioanalisis, Universidad Veracruzana, Carmen Serdan s/n. Col. Flores Magon, Veracruz, Ver., 91700. Mexico
Escuela de Medicina, Universidad Cristobal Colon, Carr. Veracruz-Medellin s/n. Col. Puente Moreno, Boca del Rio, Ver., 94271. Mexico
c
Instituto de Investigaciones Medico-Biologicas, Universidad Veracruzana, Carmen Serdan s/n. Col. Flores Maron, Veracruz, Ver., 91700.
Mexico
d
Centro Tlaxcala de Biologia de la Conducta, Universidad Autonoma de Tlaxcala, Carretera Tlaxcala-Puebla Km. 15., 90062. Mexico
Corresponding author: Alfonso Alexander Aguilera, e-mail: [email protected]
a
b
INTRODUCTION
tion as potential risk factors for metabolic syndrome and
type 2 diabetes6-8. These sugars, which include glucose are
converted into fatty acids by means of de novo lipogenesis
in enterocytes and hepatocytes, and are the source for
hepatic triglycerides synthesis9, favoring the development
of steatosis.
The development of steatosis is related to fat homeostasis in adipocytes, hepatocytes and myocytes among
other cells. This equilibrium is controlled by a number of
factors including membrane and/or intracellular proteins
implicated in the transport, synthesis and degradation
of fat10. For example, the CD36 transporter coordinates
uptake and processing of free fatty acids, as well as fatty
acids storage in the adipocytes, utilization by adipose tissues and muscle11, chylomicron production12, and secretion of hepatic VLDL (ref.13). Cd36 also contributes to
the regulation of energy balance and, therefore, has a role
in the risk of metabolic disorders such as insulin resistance, type 2 diabetes mellitus, obesity, and non alcoholic
hepatic steatosis10.
Despite current advances in understanding the complex cellular signaling of the metabolic and inflamma-
Non alcoholic fatty liver disease (NAFLD) includes
a group of hepatic diseases not caused by alcohol consumption, evolving from non alcoholic simple fatty liver
(NAFL) to non alcoholic steatohepatitis (NASH), hepatic fibrosis and cirrhosis. NAFLD has become the leading cause of chronic liver damage in developed countries1
and is a recognized condition associated with increased
cardiovascular and liver-related mortality2. This disease is
characterized by an accumulation of triglycerides (TG) in
hepatocytes known as steatosis and has been associated
with insulin resistance (IR), obesity, type 2 diabetes mellitus and dyslipidemia3. The pathogenesis of NAFLD has
been explained by the “double-hit” hypothesis, consisting
of fat or lipid accumulation as the primary insult or “first
hit” in the liver4 followed by a “second hit” in which proinflammatory mediators cause inflammation, hepatocellular injury and fibrosis5.
Causes of hepatic steatosis, include poor diet and nutrition. Sucrose or fructose intake and/or comsumption
of sugar-sweetened beverages have recently received atten99
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2018 Jun; 162(2):99-106.
tory pathways involved in NAFLD, the development of
steatosis and progression to steatohepatitis and fibrosis/
cirrhosis is not yet fully understood14,15. Some studies have
shown that the increase in CD36 expression in liver of
transgenic mice attenuates the formation of steatosis during fasting16.
The long-term consumption of sugars results in physiopathological communication between adipose and hepatic
tissues that is important in the development of steatosis
and the pathogenesis of NAFLD. Furthermore, different
cellular dynamics during the development of obesity are
associated with insulin resistance and fatty liver; among
other factors. Thus, it is important to know the relationship that exists in the transcriptional expression of the
gene encoding for the CD36 receptor under sucrose consumption in tissues associated with the development of
steatosis.
The purpose of this work was to evaluate the effects of
dietary sucrose administration and Cd36 mRNA expression levels in adipose and hepatic tissues in Wistar rats
on the development of non-alcoholic fatty liver disease
(NAFLD).
MATERIALS AND METHODS
Experimental design
Twenty Wistar rats, all nearly 21 days old, were purchased from Harlan Teklad Inc. (Mexico City). The animals, were individually housed in stainless-steel cages and
maintained in 12-h light/dark cycles at 25 °C. Animal
maintenance and handling followed the guidelines of
Mexican legislation, NOM-062-ZOO-1999 (ref.17).
Animals were divided into two experimental groups:
two non-alcoholic fatty liver groups (NAFL-10 Group, n-5
and NAFL-20 Group, n-5) that received a standard diet
(2014 Teklad Global) plus 30% sucrose in their drinking
water for 10 or 20 weeks, and a control groups (C Groups)
that received a standard diet and drinking water.
After experimental treatment with sucrose; fasted
animals (18 hrs) from each group were sacrificed.
Measurements of body weight, food intake, and serum
parameters were made. Adipose and hepatic tissues were
extirpated some tissue was preserved in formalin and
some tissue at -70 ° C until analysis.
Serum biochemical determinations
Serum glucose, cholesterol, triglycerides, uric acid,
alanine aminotransferase (ALT), aspartate aminotransferase (AST) and other markers, were determined by
enzymatic methods using a Selectra-E autoanalyzer. All
reagents were obtained from Spinreact SA in Spain. The
serum insulin concentration was determined by a commercial double-antibody solid-phase radioimmunoassay
(Coat-A-Count, DPC). Serum free fatty acids (FFAs)
were determined from fresh frozen samples by an enzymatic method (NEFA-C test, Wako Chemicals). To
calculate the index of insulin resistance (HOMA-IR) the
following formula was used: insulin [µU/mL] x glucose
[mmol/L]/ 22.5 (ref.18).
100
Quantification of Hepatic Triglycerides
Lipids were extracted from hepatic tissue (1 g) utilizing the method of Folch et al.19. Total lipids were extracted
from liver samples by homogenizing the tissues using a
mixture of chloroform/methanol/0.9% NaCl to a final 20fold dilution of the original volume of the tissue sample.
The organic layer was then removed, evaporated, and
reconstituted in chloroform. The values of TG were measured using a colorimetric assay kit (Spinreact, Barcelona,
Spain) and the results were expressed as mg per gram of
the liver weight.
Adipocyte size measurements
Random samples of abdominal adipose tissue of rats
in the NAFLD and control group were fixed in neutral
formalin (10% formaldehyde and 0.1 M phosphate buffer,
pH 7) for 24 h at room temperature. The samples were
embedded in paraffin, and 7 mm serial sections were then
cut using a microtome and stained with hematoxylin and
eosin.
Areas were measured in each adipocytes for microscopic field of view, using five fields of view per rat. The
average adipocyte area was calculated for each rat, and
group means were determined from the individual averages for comparisons between groups. For the above,
photomicrographs were obtained at a 40X magnification
utilizing an optical microscope (Axio Image A2, Zeiss)
with an Olympus digital camera at a 5.1-megapixel resolution. Adipocyte areas were measured using the AxioVision
Real 4.6 software (Zeiss Software, Inc.), and expressed
as µm2.
Microscopic liver analysis
To analyze hepatic damage, liver tissue was fixed for
24 h in 10% neutral buffered formalin. Sections (4-6 µm)
of the tissue were embedded in paraffin, and stained
with hematoxylin and eosin (H–E) prior to microscopic examination. Digital images were obtained using an
Olympus BX51 microscope equipped with a Camedia
C3040ZOOM digital camera (Olympus America Inc.,
Corporate Center Drive, Melville, NY, USA). All the images were recorded at 10x and 40x magnification.
Real-time PCR Cd36 Gene
a) Total RNA Isolation
Total RNA was obtained from nitrogen-frozen rat liver
or abdominal fat tissue. First 300 mg of nitrogen-frozen
tissue was pulverized using a nitrogen-cooled steel mortar. The tissue powder, was immediately suspended in 2.0
mL of TRIzol™ Reagent (Invitrogen) and incubated at
room temperature 5 min. Then, 0.3 mL of chloroform was
added and shaken for 15 s, and after three minutes the
samples were centrifuged at 12,000 x g for 15 min at 4 °C
and the aqueous phase was recovered. The RNA was precipitated with 1 mL of isopropanol and incubated at room
temperature for 10 min and recovered by centrifugation at
12,000 x g for 20 min at 4 °C. The RNA was washed with
ethanol at 75%, and resuspended in RNase-free water and
kept at -70 °C until use.
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2018 Jun; 162(2):99-106.
Table 1. Caloric consumption parameters, fat and liver weight in sucrose-fed NAFLD and Control groups.
Initial body weight (g)
Final body weight (g)
Liquid intake (mL/day)
Liquid intake (mL/day/100 g bw)
Equivalent in Kcal in drinking water
Food intake (g/day)
Food intake (g/day/100 g bw)
Equivalent in Kcal in food
Total Kcal/day/100 g bw
Abdominal fat weight (g)
Epididymal fat weight (g)
Total fat (g)
Number of adipocyte x 106 (mm2)
Liver weight (g)
Hepatic triglycerides (mg/g liver)
Index of adiposity
Hepatosomatic index
10 Weeks
Control Group
NAFLD-10 Group
172±8
186±7
341±50
379±44
85.43±11.01
145.30±21.35*
24.99±3.22
38.31±5.62*
0.00
45.96±6.74*
58.67±8.42
28.22±4.00*
17.16±2.46
7.44±1.05*
53.19±7.58
23.06±3.25*
53.19±7.58
69.02±9.99
9.20±1.06
15.93±3.95*
7.20±1.40
15.10±2.20*
16.40±2.46
31.03±6.15*
29.33±3.70
31.34±3.80
9.76±0.20
13.30±0.26*
15±2.50
22±1.85*
4.80
8.18*
2.86
3.50
20 Weeks
Control Group
NAFLD-20 Group
172±8
186±7
359±49
394±47
112.47±13.20
209.91±17.24*
31.32±3.67
53.27±4.37*
0.00
63.92±1.31*
61.76±1.14
12.80±4.63*
17.89±2.35
3.24±1.17*
55.45±7.28
10.04±3.62*
55.45±7.28
73.96±4.93*
10.76±1.37
16.90±2.72*
8.75±2.31
17.30±1.96*
19.51±3.68
34.20±4.68*
38.80±5.80
40.60±4.60
9.90±0.83
13.32±0.71*
17±2.80
35±1.80*
5.43
8.68*
2.75
3.38
Values are the mean ± SD (C, n=5; NAFLD-10 and NAFLD-20, n=5). * P<0.01. Bw = Body weight. Index of adiposity= (Abdominal and epididymal fat weight / body weight) x 100. Hepatosomatic index = (liver weight/body weight) x 100
of five repetitions. The Minitab 16.1.0 statistical package
was used.
RESULTS
Caloric intake from diet, weight and body fat
A model of fatty liver was achieved by the administration of 30% sucrose in drinking water in male Wistar rats.
Figure 1 shows the evolution of body weight of the rats
that consumed sucrose over 20 weeks, the NAFLD-10
and NAFLD-20 groups consuming sucrose for 10 and
20 weeks showed no significant changes in body weight
450
*
400
Body weight (g)
b) Cd36 mRNA expression measurement
The relative expression of Cd36 mRNA from abdominal fat and hepatic tissue was measured with the
StepOne™ Real Time PCR System (Life Technologies).
The reaction was performed with iTaq™ Universal
SYBR® Green One-Step Kit (Bio-Rad). The amount
and quality of total RNA were determined by spectrophotometry using NanoDrop 2000™ spectrophotometer
(Thermo Scientific) and 0.5 µg for each sample of total
RNA was normalized for each reaction. The reaction
mix was adjusted according to the manufacturer’s recommendations; primers used for Cd36 gene expression
were reported by Arias et al., 2014 (sense 5’-GGT GTG
CTC AACAGC CTT ATC-3’and antisense 5’-TTA TGG
CAA CCTTGC TTA TG-3’) (ref.20). Thermocycling
conditions were as follows: for reverse transcription ten
minutes at 50°C, for polymerase activation and DNA denaturation one minute at 95 °C and for amplification,
40 cycles of 15 s at 95 °C and 60 s at 60 °C. β-Actin
gene expression was used as the endogenous control, the
primers sequences used for β-Actin were as follows: sense
5'-TGGAATCCTGTGGCATCCATG-3 and antisense
5'-TAAAACGCAGCTCAGTAACAG-3’. Melting curve
analysis were performed after each RT-PCR run in order
to confirm that only one PCR products was detected.
The relative expression of the target gene with respect to
the internal control was calculated using relative quantification software provided by StepOne™ Real Time PCR
System.
350
*
300
Control Group
250
NAFLD Group
200
150
1
5
10
15
20
Data analysis
Weeks
Experimental data were tested for normality and homogeneity of variance. We then used ANOVA and com- Fig. 1. Evolution of the body weight of rats that consumed
forbody
10 weeks
(NAFLD-10
Group)
and 20sucrose
weeks for 10 wee
parisons using the multiple range Tukey testFig.
(P<0.05
or sucrose
1. Evolution
of the
weight
of rats that
consumed
(NAFLD-20 Group) with respect to control rats.
P<0.01); the results are expressed as the means
and
SDs
10 Group) and 20 weeks (NAFLD-20 Group) with respect to control rats.
101
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2018 Jun; 162(2):99-106.
A
100
Control
Group
NAFLD-10
Group
Porcentages %
75
50
*
25
*
*
0
0-500
501-1000
2
1001-1500
>1501
µm
B
100
Control
Group
NAFLD-20
Group
75
Porcentages %
compared to the control groups. However, there were significant changes in weight at weeks 5 and 15 of consuming sucrose.
Table 1 shows the differences in liquid consumption
as well as food and caloric intake at the end of the sucrose treatment period at 10 or 20 weeks (P<0.01). At 10
weeks, the ingestion of sucrose in NAFLD-10 group led
to a significant increase in abdominal fat of 73% (P<0.01)
and epididymal fat of 109% (P<0.01). After 20 weeks of
sucrose administration, the rats in the NAFLD-20 group
presented an increase in abdominal fat of 57%, (P<0.01)
and epididymal fat of 97% (P<0.01) in comparison to control groups. The rats in the NAFLD 10 and 20 groups,
also showed an increase in adiposity index values (70%
and 59%; respectively) with respect to the control group
(P<0.01). Additionally, the rats in the NAFLD 10 and 20
groups presented a significant increase in liver weight and
in the quantified triglycerides in the liver (46% and 105%
respectively) in comparison to control group (P<0.01).
Serum biochemical parameters
**
50
In relation to the serum parameters, the rats in the
NAFLD-10 group consuming sucrose for 10 weeks
*
showed elevation of triglycerides (48%, P<0.05), VLDL
25
(41%, P<0.01), fatty free acids (30%, P<0.05), uric acid
*
(37%, P<0.05), insulin (85%, P<0.05) and HOMA-IR in0
dex (99%, P<0.05). Similarly, the rats consuming sucrose
0-500
501-1000
1001-1500
>1501
2
for 20 weeks (NAFLD-20 group) showed an increase in
µm
triglycerides (109%, P<0.05), VLDL (112%, P<0.01), free
Fig. 2.ofDistribution
adipocyte
rats with abdominal
fatty acids (17%, P<0.01), uric acid (18%, P<0.05)
Fig.insulin
2. Distribution
adipocyte of
sizes
in rats sizes
with inabdominal
obesity and non-alcohol
obesity
and
non-alcoholic
fatty
liver
disease
(NAFLD).
(57%, P<0.05), HOMA-IR index (52%, P<0.05)fatty
andliver
ALTdisease
(NAFLD).
(610%, P<0.05) with respect to the control group. No dif- Percentage of adipocytes between 500 and more than 1501 µm2.
2
rats consuming
sucrose
weeksthan
(NAFLD-10)
(B) rats consumin
Percentage
adipocytes
between
500 for
and10more
1501 µmand
. (A)
ference was observed in the levels of cholesterol,
total of (A)
rats
consuming
sucrose
for
20
weeks
(NAFLD-20);
relative
toweeks (NAFLD
sucrose
for
10
weeks
(NAFLD-10)
and
(B)
rats
consuming
sucrose
for
20
proteins, albumin and creatinine in rats of the NAFLD
control
rats.
*P<0.05
20);
relative
to
control
rats.
*P<0.05
groups in comparison to the control group (Table 2).
Table 2. Serum parameters in control (C) and sucrose-fed (NAFL) rats.
Glucose (mg/dL)
Insulin (µUI/mL)
HOMA-IR
Triglycerides (mg/dL)
Free fatty acids (mmol/L)
Cholesterol (mg/dL)
HDL (mg/dL)
LDL (mg/dL)
VLDL (mg/dL)
Total Proteins (g/L)
Albumin (g/L)
AST (U/L)
ALT (U/L)
Uric acid (mg/dL)
Creatinine (mg/dL)
Urea (mg/dL)
Control Group
145.00±4.50
5.79±0.44
2.14±0.01
64.00±3.46
0.68±0.04
109.67±10.26
28.00±3.00
68.67±5.00
13.00±1.73
6.60±0.10
3.80±0.20
41.50±0.55
28.00±1.00
0.80±0.01
0.42±0.08
43.00±10.19
10 Weeks
NAFLD-10 Group
161.00±7.00
10.76±1.91*
4.27±0.03*
95.50±2.50*
0.89±0.06*
135.00±13.50*
40.00±8.00*
75.90±1.00*
19.10±0.57*
7.23±0.66
3.83±0.35
26.67±2.57*
38.07±4.24*
1.10±0.01*
0.40±0.09
27.00±0.01*
Values are the mean ± SD (C, n=5; NAFLD-10, n=5) and (C, n=5; NAFLD-20, n=5).
* P<0.05
102
20 Weeks
Control Group
NAFLD-20 Group
156.22±11.72
151.10±6.43
7.53±0.66
11.85±1.45*
2.90± 0.01
4.41±0.02*
82.13±3.44
170.40±25.00*
0.80±0.04
0.94±0.03*
92.16±1.40
83.30±6.60
24.32±5.20
22.74±6.82
51.42±6.66
26.48± 4.00*
16.42±0.68
34.08±9.40*
6.47±0.17
6.64±0.25
4.02±0.08
4.18±0.29
37.27±1.20
48.40±1.75*
33.15±2.17
235.50±2.89*
0.86±0.05
1.02±0.01*
0.50±0.08
0.49±0.09
40.16±3.85
19.72±4.31*
1
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2018 Jun; 162(2):99-106.
Cellular dynamic of adipose tissue
Fig. 2. shows the relative distribution of adipocyte areas in abdominal adipose tissue samples from rats in the
NAFLD-10 and NAFL-20 groups that consumed sucrose.
The NAFLD-10 group displayed a significant increase in
the percentage of fat cells with areas between 1001 and
1501 μm2 compared with the control group that presented
fat cells smaller than 1000 µm2 (P<0.01). In the same way,
the NAFLD-20 group showed a significant increase in the
percentage of fat cells with areas greater 1001 µm2 and
a reduction of adipocytes with areas between 501-1000
µm2 compared to the control group (P<0.05). The adipose
tissue of rats fed sucrose showed greater adipocyte hypertrophy than the control animals (Fig. 3).
Control Groups
NAFLD Groups
At 10
weeks
(a)
(b)
At 20
weeks
Microscopic liver analysis
After 10 weeks of sucrose administration, the rats in
the NAFLD-10 group showed an increase in liver weight.
(d)
(c)
However, there were no changes in the hepatosomatic
index. Similarly, at 20 weeks of consuming sucrose, the
rats in the NAFLD-20 group presented an increase in liver Fig. 3. Abdominal adipose tissue of rats in the control groups
consuming water without sucrose (a, c) and hypertrophic
weight, but no elevation in the hepatosomatic index, Fig.
with3. Abdominal
adipose tissue of rats in the control groups consuming water
adipose tissue in rats consuming sucrose for 10 weeks in
respect to the control group.
sucrose (a, c) and hypertrophic adipose tissue in rats consuming sucrose for 10
NAFLD-10 group (b) and 20 weeks in NAFLD-20 group (d).
The microscopic liver analysis of rats in the NAFLD-10
NAFLD-10 group (b) and 20 weeks in NAFLD-20 group (d).
group showed the presence of mild portal hepatitis (Fig.
4b and c); while the rats in NAFLD-20 group had grade
Fig. 4. Histological analysis of the liver in rats in the NAFLD-10 group consuming sucrose for 10
weeks: (a) normal hepatic parenchyma (10×) in control group, (b) scarce mononuclear inflammatory infiltrate in the stroma of portal spaces (10×) and (c) hepatocytes without alterations (mild
Fig.
Histological
analysis
liver in rats
in the
NAFLD-10
group
consuming
portal4.hepatitis)
(40×).
Rats inof
thethe
NAFLD-20
group
consuming
sucrose
for 20
weeks: (d)sucrose
normal
for
10 parenchyma
weeks: (a)(10×)
normal
hepatic
parenchyma
(10x) ininthecontrol
group,
(b) in
scarce
hepatic
in control
group;
(e) lymphocytes
portal space
stroma,
addimononuclear
inflammatory
in theandstroma
of portal detail
spaces
(10x) and with
(c)
tion hepatocytes
with large lipidinfiltrate
vacuoles (10×)
(f) morphological
of hepatocytes
large lipid vacuoles
grade
1) (40×).
and Eosin.
hepatocytes
without(steatosis
alterations
(mild
portalHematoxylin
hepatitis) (40x).
Rats in the NAFLD-20 group
consuming sucrose for 20 weeks: (d) normal hepatic parenchyma (10x) in control group;
(e) lymphocytes in the portal space stroma, in addition hepatocytes with large lipid
103
vacuoles (10x) and (f) morphological detail of hepatocytes with large lipid vacuoles
(steatosis grade 1) (40x). Hematoxylin and Eosin.
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2018 Jun; 162(2):99-106.
DISCUSSION
1 hepatic steatosis with mild portal inflammation, as described by Brunt et al., 1999 (Fig. 4e and f). The control
rats showed a normal histology of the hepatic parenchyma
(Fig. 4a and c) (ref.21).
The histological analysis showed the development of
mild portal hepatitis in rats of the NAFLD-10 group and
grade 1 hepatic steatosis with mild portal inflammation
in rats of the NAFLD-20 group.
Expression of Cd36 in adipose and hepatic tissues
The Cd36 mRNA levels were different in adipose and
hepatic tissues after 10 and 20 weeks of administration
of sucrose in drinking water (ad libitum) in Wistar rats
with fatty liver (Fig. 5). The rats in the NAFLD-10 group
showed significantly higher (1.5-fold; P<0.05) levels of
Cd36 mRNA in hepatic tissue in comparison to the control group (Figure 5B), whereas the expression levels in
adipose tissue remained unchanged (Fig. 5A). The rats
in the NAFLD-20 group had even higher levels of Cd36
mRNA in hepatic tissue (4.5 fold; P<0.05) with respect
to control rats (Fig. 5D), while in adipose tissue the expression levels remained the same in comparison to rats
without sucrose intake (Fig. 5C).
4,5
Cd36 mRNA exprssion
B
5
4
4,5
3,5
3
2,5
2
1,5
1
0,5
Control Group
4
3,5
*
3
2,5
2
1,5
1
0
NAFLD-10
Group
Control Group
D
Abdominal fat
Cd36 mRNA expression
Cd36 mRNA expression
5
4,5
4
3,5
3
2,5
2
1,5
1
0,5
0
Liver
0,5
0
C
5
Abdominal fat
Cd36 mRNA expression
A
The objective of this work was to evaluate the effects
of sucrose on the development of fatty liver as well as on
Cd36 mRNA expression in adipose and hepatic tissues
in Wistar rats.
The consumption of fructose, as sucrose or as highfructose corn syrup (HFCS), has increased drastically in
recent years along with increasing rates of obesity, type
2 diabetes, hypertension, kidney damage22 and metabolic
syndrome, which are risk factors for the development of
NAFLD.
Studies have reported on the role of the CD36 protein in the development of NAFLD and other chronicdegenerative diseases such as hypertension and metabolic
syndrome. However, depending on the study model, there
are differences in the levels of Cd36 gene expression23,24;
which can remain unchanged or increase in the tissues.
The Cd36 mRNA expression is the first molecular
mechanism associated to development of fatty liver, for
this reason it is very important to learn how Cd36 gene
expression in the liver, is affected by different risk factors
including the diet.
Control Group
NAFLD-20
Group
5
4,5
4
3,5
3
2,5
2
1,5
1
0,5
0
Liver
Control Group
NAFLD-10
Group
**
NAFLD-20
Group
Fig. 5. Expression levels of Cd36 mRNA in adipose and hepatic tissues of rats consuming sucrose (NAFLD-10
and 20 groups) with respect to control rats. At 10 weeks, adipose (A) and hepatic tissue (B). At 20 weeks, adipose
(C)Fig.
and hepatic
tissues (D).levels
*P<0.05;
versus control.
5. Expression
of **P<0.01
Cd36 mRNA
in adipose and hepatic tissues of rats consuming
sucrose (NAFLD-10 and 20 groups) with respect to control rats. At 10 weeks, adipose (A)
104
and hepatic tissue (B). At 20 weeks, adipose
(C) and hepatic tissues (D). *P<0.05;
**P<0.01 versus control.
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2018 Jun; 162(2):99-106.
After 10 weeks of ingesting sucrose in drinking water,
rats of the NAFLD-10 group showed no significant changes in total calorie consumption with respect to the control
group; however, at 20 weeks of ingesting sucrose, the rats
in NAFLD-20 group showed an increase in total calorie
ingestion compared to control rats. These dietary calories
were not reflected in the body weight of the rats of the
NAFLD-10 and 20 groups, however; the rats presented
significant increased abdominal and epididymal fat.
These results coincide with other reports which have
shown that consumption of sugary drinks in humans and
animals is associated with obesity, cardiovascular diseases
and metabolic syndrome7,25, as well as increased ectopic
fat accumulation, regardless of other lifestyle factors26.
The above results are related to the serum biomarkers,
which showed significant changes in the rats consuming
sucrose with respect to the control group. At 10 and 20
weeks of sucrose intake, the rats of the NAFLD groups
showed an increase in triglycerides, VLDL, free fatty acids, insulin, HOMA-IR and uric acid. The rats consuming
sucrose showed a significative increase in total body fat
which is associated with dyslipidemia (elevation of triglycerides and VLDL) and insulin resistance. These results
are similar to those of other research groups who have
shown that the accumulation of abdominal fat results in
excessive liberation of fatty acids from the visceral adipose
tissue, increasing the risk of hyperinsulinemia and insulin
resistance which are associated with an inflammatory and
thrombogenic profile27-29.
The rats in the NAFLD groups that consumed sucrose (NAFLD-10 and NAFLD-20) showed similar cellular dynamics in adipose tissue to those reported by Jo
et al., who studied adipocyte size and distribution in several degenerative diseases30,31, as well as examining how
adipocyte population size shifts under various dietary
conditions and among different rat strains. The increase
in the percentage of adipocytes with a size greater than
1000 µm2 in rats that consumed sucrose agree with other
studies that show that the size distribution differences
obtained correlate with a diabetic phenotype32. This is
characterized by hypertrophy of adipocyte associated with
hepatic steatosis development and inflammation33.
In connection with the Cd36 gene expression and development of fatty liver in rats, we have shown that the
increase in triglycerides and fatty acids in the sera of rats
in the NAFLD-10 and NAFLD-20 groups could be associated with the flux of free fatty acids from adipose tissue
to liver as well as the development of grade 1 hepatic
steatosis with mild portal inflammation after 20 weeks
of sucrose consumption. The rats with fatty liver showed
significant differences in Cd36 gene expression; while the
expression levels in adipose tissue remained unchanged
relative to control rats. The liver showed increased levels
of Cd36 gene expression between weeks 10 and 20 of
sucrose administration. These results indicate that hypertrophy and the storage capacity of adipose tissue allow
it to maintain its basal levels of Cd36 expression. Since
the liver is an organ that cannot increase in size unlike
adipose tissue, it is forced to raise expression levels of the
Cd36 gene. This could lead to in synthesis of the recep105
tor protein, as one of the molecular mechanisms for the
development of NAFLD.
Some authors have shown that insulin resistance increases the expression of the Cd36 gene in rat liver in
a PPARγ-dependent manner, by increasing the localization of the CD36 receptor in the plasma membrane of
the hepatocyte, promoting the development of NAFLD
(ref.34-36). Additionally, there are reports of CD36 receptor
participation in the development of NAFLD associated
with aging in animals and humans. It has been shown that
aging increase the circulating levels of insulin, glucose and
fatty acids in humans and mice37. Factors have been documented to either increase cellular CD36 expression or induce CD36 translocation to the plasma membrane38,39,40,41,
increasing the risk of NAFLD. Our results demonstrate
that intake of sucrose could promote cellular conditions
that increase the Cd36 mRNA expression.
CONCLUSION
In conclusion, sucrose intake for 10 and 20 weeks by
Wistar rats caused abdominal obesity, insulin resistance
and development of NAFLD, along with a significant
increase in the expression of the Cd36 gene in hepatic
tissue, but not in adipose tissue.
Author contribution: All authors contributed equally to
preparing the manuscript.
Conflict of interest statement: None declared.
REFERENCES
1. Romeo GR, Lee J, Shoelson SE. Metabolic syndrome, insulin resistance, and roles of inflammation–mechanisms and therapeutic
targets. Arterioscler Thromb Vasc Biol 2012;32:1771-6.
2. Angulo P.Obesity and nonalcoholic fatty liver disease. Nutr Rev
2007;65:57-63.
3. Marchesini G, Bugianesi E, Forlani G, Cerrelli F, Lenzi M, Manini R,
Natale S, Vanni E, Villanova N, Melchionda N, Rizzeto M. Nonalcoholic
fatty liver, steatohepatitis, and the metabolic syndrome. Hepatology
2003;37:917-23.
4. Farrell GC, Larter CZ. Nonalcoholic fatty liver disease: From steatosis
to cirrhosis. Hepatology 2006;43:S99-S112.
5. Tacke F, Luedde T, Trautwein C.Inflammatory pathways in liver homeostasis and liver injury. Clin Rev Allergy Immunol 2009;36:4-12.
6. Johnson L, Mander AP, Jones LR, Emmett PM, Jebb, SA. Is sugarsweetened beverage consumption associated with increased fatness
in children? Nutrition 2007;23:557-63.
7. Palmer JR, Boggs DA, Krishnan S, Hu FB, Singer M, Rosenberg L.
Sugar-sweetened beverages and incidence of type 2 diabetes mellitus in African American women. Arch Intern Med 2008;168:1487-92.
8. Segal, M.S., Gollub, E., Johnson, R.J. Is the fructose index more relevant with regards to cardiovascular disease than the glycemic index? Eur J Nutr 2007;46:406-17.
9. Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt MD, Parks
EJ. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest
2005;115:1343-51.
10. Silverstein RL, Febbraio, M. CD36, a scavenger receptor involved
in immunity, metabolism, angiogenesis, and behavior. Sci Signal
2009;2:re3.
11. Pepino MY, Kuda O, Samovski D, Abumrad NA. Structure-function
of CD36 and importance of fatty acid signal transduction in fat metabolism. Annu Rev Nutr 2014;34:281-303.
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2018 Jun; 162(2):99-106.
12. Tran TT, Poirier H, Clement L, Nassir F, Pelsers MM,Petit V, Degrace P,
Monnot MC, Glatz JF, Abumrad NA, Besnard P, Niot I. Luminal lipid
regulates CD36 levels and downstream signaling to stimulate chylomicron synthesis. J Biol Chem 2011;286:25201-10.
13. Nassir F, Adewole OL, Brunt EM, Abumrad NA. CD36 deletion reduces
VLDL secretion, modulates liver prostaglandins, and exacerbates
hepatic steatosis in ob/ob mice. J Lipid Res 2013;54:2988-97.
14. Berlanga A, Guiu-Jurado E, Porras JA, Auguet T. Molecular pathways in non-alcoholic fatty liver disease. Clin Exp Gastroenterol
2014;7:221-39.
15. Tilg H, Moschen AR. Evolution of inflammation in nonalcoholic fatty liver disease: The multiple parallel hits hypothesis. Hepatology
2010;52:1836-46.
16. Garbacz WG, Lu P, Miller TM, Poloyac SM, Eyre NS, Mayrhofer G, Xu
M, Ren S, Xie W. Hepatic overexpression of CD36 improves glycogen
homeostasis and attenuates high-fat diet-induced hepatic steatosis
and insulin resistance. Mol Cell Biol 2016;36(21):2715-27.
17. SAGARPA. Norma Oficial Mexicana NOM-062-ZOO-1999,
Especificaciones técnicas para la producción, cuidado y uso de los
animales de laboratorio. Diario Oficial de la Federación. Fecha de
publicación 22 de agosto de 2001.
18. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner
RC. Homeostasis model assessment: insulin resistance and beta-cell
function from fasting plasma glucose and insulin concentrations in
man. Diabetologia 1985;28:412-9.
19. Folch J, Lees M, Sloane Stanley G.H, A simple method for the isolation and purification of total lipides from animal tissues. J Biol
Chem 1957;226:497-509.
20. Arias N, Macarulla MT, Aguirre L, MartÍnez-Castaño MG, Portillo MP.
Quercetin can reduce insulin resistance without decreasing adipose
tissue and skeletal muscle fat accumulation. Genes Nutr 2014;9:361.
21. Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Bacon
BR. Nonalcoholic steatohepatitis: a proposal for grading and staging
the histological lesions. Am J Gastroenterol 1999;94:2467-74.
22. Johnson, RJ, Segal MS, Sautin Y, Nakagawa T, Feig DI, Kang DH,
Gersch MS, Benner S, Sanchez-Lozada LG. Potential role of sugar
(fructose) in the epidemic of hypertension, obesity and the metabolic syndrome, diabetes, kidney disease, and cardiovascular disease. Am J Clin Nutr 2007;86:899-906.
23. Aitman TJ, Glazier AM, Wallace CA, Cooper LD, Norsworthy PJ, Wahid
FN, Al-Majali KM, Trembling PM, Mann CJ, Shoulders CC, Graf D, St
Lezin E, Kurtz TW, Kren V, Pravenec M, Ibrahimi A, Abumrad NA,
Stanton LW, Scott J. Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in
hypertensive rats. Nat Genet 1999;21(1):76-83.
24. Alexander AA, Angulo GO, Quintana CR, Ida Soto RI, Guadalupe
Sánchez OMG, Rosa María Oliart RRM. CD36 gene expression induced by fish oil in abdominal adipose tissue of rats with metabolic
syndrome. J Food Nutr Disord 2017;6(2):1-6
25. Dhingra R, Sullivan L, Jacques PF, Wang TJ, Fox CS, Meigs JB,
D’Agostino RB, Gaziano JM, Vasan RS. Soft drink consumption and
risk of developing cardiometabolic risk factors and the metabolic
syndrome in middle-aged adults in the community. Circulation
2007;116:480-8.
26. Nseir W, Nassar F, Assy N. Soft drinks consumption and nonalcoholic
fatty liver disease. World J Gastroenterol 2010;16:2579-88.
27. Desprees JP, Lemieux I, Bergeron J, Pibarot P, Mathieu P. Larose E,
Rodés Cabau J, Bertrand OF, Poirier P. Abdominal obesity and the
106
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
metabolic syndrome: contribution to global cardiometabolic risk.
Arterioscler Thromb Vasc Biol 2008;28:1039-49.
Tsuriya D, Morita H, Morioka T, Takahashi N, Ito T, Oki Y, Nakamura
H. Significant correlation between visceral adiposity and high-sensitivity C-reative protein (hs-CRP) in Japanese subjects. Intern Med
2011;50:2767-73.
Reyes M, Gahagan S, Díaz E, Blanco E, Leiva L, Lera L, Burrows R.
Relationship of Adiposity and insulin Resistance Mediated by inflammation in a Group of Overweight and Obese Chilean Adolescents.
Nutr J 2011;10:1-4.
Jo J, Gavrilova O, Pack S, Jou W, Mullen S, Sumner AE, Cushman SW,
Periwal V. Hypertrophy and/or hyperplasia: dynamics of adipose
tissue growth. PLoS Comput Biol 2009;3:e1000324.
Jo J, Guo J, Liu T, Mullen S, Hall KD, Cushman SW et al. HypertrophyDriven adipocyte death overwhelms recruitment under prolonged
weight gain. Biophys J 2010;11:3535-44.
Fang L, Guo F, Zhou L, Stahl R, Grams J. The cell size and distribution
of adipocytes from subcutaneous and visceral fat is associated with
type 2 diabetes mellitus in humans. Adipocyte 2015;4:273-9.
Kursawe R, Eszinger M, Narayan D, Liu T, Bazuine M, Cali AMG,
D’Adamo E, Shaw M, Pierpont B, Shulman GL, Cushman SW, Sherman
A, Caprio S. Cellularity and adipogenic profile of the abdominal subcutaneous adipose tissue from obese adolescents: association with
insulin resistance and hepatic steatosis. Diabetes 2010;9:2288-96.
Lee YJ, Ko E H, Kim JE, Kim E, Lee H, Choi H, Yu JH, Kim HJ, Seong, JK,
Kim KS, Kim JW. Nuclear receptor PPAR- regulated monoacylglycerol
O-acyltransferase 1 (MGAT1) expression is responsible for the lipid
accumulation in diet-induced hepatic steatosis. Proc Natl Acad Sci
USA 2012;109:13656-61.
Rahimian R, Masih-Khan E, Lo M, van Breemen C, McManus BM,
Dubé GP. Hepatic overexpression of peroxisome proliferator activated receptor γ2 in the ob/ob mouse model of non-insulin dependent diabetes mellitus. Mol Cell Biochem 2001;224:29-37.
Schadinger SE, Bucher NL, Schreiber BM and Farmer SR. Insulin
Enhances Hepatic Expression of FA Translocase CD36 PPARγ2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes.
Am J Physiol Endocrinol Metab 2005;288:E1195-E1205.
Sheedfar F, Sung MM, Aparicio-Vergara M, Kloosterhuis NJ,
Miquilena-Colina ME, Vargas-Castrillón J, Febbraio M, Jacobs RL,
de Bruin A, Vinciguerra M, García-Monzón C, Hofker MH, Dyck JR,
Koonen DP. Increased hepatic CD36 expression with age is associated with enhanced susceptibility to nonalcoholic fatty liver disease.
Aging (Albany NY) 2014;6:281-95.
Chang AM, Halter JB. Aging and insulin secretion. Am J Physiol
Endocrinol Metab 2003;284:E7-12.
Bonen A, Benton CR, Campbell SE, Chabowski A, Clarke DC, Han XX,
Glatz JFC, Luiken JJFP. Plasmalemmal fatty acid transport is regulated in heart and skeletal muscle by contraction, insulin and leptin,
and in obesity and diabetes. Acta Physiol Scand 2003;178:347-56.
Coort SLM, Hasselbaink DM, Koonen DPY, Willems J, Coumans
WA, Chabowski A, van der Vusse GJ, Bonen A, Glatz JFC, Luiken
JJFP. Enhanced sarcolemmal FAT/CD36 content and triacylglycerol storage in cardiac myocytes from obese zucker rats. Diabetes
2004;53:1655-63.
Schwenk RW, Luiken JJFP, Bonen A, Glatz JFC. Regulation of sarcolemmal glucose and fatty acid transporters in cardiac disease.
Cardiovasc Res 2008;79:249-58.